Abstract:

Methods of using probes and probe sets for the detection of high grade
dysplasia and carcinoma in cervical cells are described. Methods of the
invention include hybridizing one or more chromosomal probes to a
biological sample obtained from a subject and detecting the hybridization
pattern of the chromosomal probes to the sample to determine whether the
subject has high grade dysplasia or carcinoma. Methods of the invention
also include preliminary screening the cells for a marker associated with
a risk for cancer, and preferably involves screening for HPV infected
cells by in situ hybridization using an HPV probe mixture.

Claims:

1. A method for screening for cervical high grade squamous intraepithelial
lesion HSIL in a human subject, the method comprising:a. obtaining a
cervical sample containing a plurality of cervical cells from the
subject;b. contacting the sample with a set of two or more chromosomal
probes comprising chromosomal probes specific for loci 8q24 and 20q13
able to detect HSIL under conditions sufficient to enable hybridization
of the probes to any chromosomes in the sample; and,c. detecting the
hybridization pattern of the chromosomal probes to the plurality of cells
in the sample, wherein said hybridization pattern is indicative of
amplification of loci 3q26 and 8q24 and correlating said amplification of
loci 3q26 and 8q24 with the presence of cervical HSIL in said subject.

4. The method of claim 1, wherein the chromosomal probes are fluorescently
labeled.

5. The method of claim 1, wherein the set of two or more chromosomal
probes additionally comprises probes specific for 3q26, Xp22 and CEP 15.

6. The method of claim 1, wherein the set of two or more chromosomal
probes comprises probes specific for 3p21, 3 p14 and CEP 3.

7. The method of claim 1 wherein cells from the cervical sample are
prescreened for a marker associated with risk for cancer.

8. The method of claim 1 wherein cells from the cervical sample are
prescreened for infection by HPV.

9. The method of claim 8 wherein the sample is screened for infection by
one or more of the high risk HPV types selected from the group consisting
of 16, 18, 31, 33, 35, 52 and 58.

10. The method of claim 1 wherein cells from the cervical sample are
prescreened for the presence of a cell cycle protein or a cell
proliferation marker.

11. The method of claim 10 wherein the cell cycle protein is p16 or Cyclin
E.

12. The method of claim 10 wherein the cell proliferation marker is the
protein Ki67 or the protein PCNA.

13. A method for screening for cervical HSIL in a human subject, the
method comprising:a. obtaining a cervical sample containing a plurality
of cervical cells from the subject;b. contacting the sample with a set of
two or more chromosomal probes comprising chromosomal probes specific for
loci 8q24 and 20q13 able to detect HSIL and a mixture of HPV probes under
conditions sufficient to enable hybridization of the probes to any
chromosomes in the sample and sufficient to enable detection of any HPV
infected cells present in the sample;c. detecting the presence of HPV
infected cells in the sample;d. determining hybridization pattern of the
chromosomal probes in the HPV infected cells in the plurality of cervical
cells in the sample, wherein said hybridization pattern is indicative of
amplification of loci 3q26 and 8q24 and correlating said amplification of
loci 3q26 and 8q24 with the presence of cervical HSIL.

14. The method of claim 13 wherein the mixture of HPV probes consists of
probes substantially complementary to full coding sequence for each of
HPV-16, HPV-18, HPV-30 and HPV-58.

15. The method of claim 13, wherein the set of two or more chromosomal
probes additionally comprises probes specific for 3q26, Xp22 and CEP 15.

16. The method of claim 1, wherein the set of two or more chromosomal
probes comprises probes specific for 3p21, 3p14 and CEP 3.

17. The method of claim 15 wherein the set of two or more chromosomal
probes comprises probes to the TERC locus at 3q26, the cmyc locus at 8q24
and the centromere of chromosome 8.

18. The method of claim 14 wherein hybridization conditions are sufficient
to detect the presence of any of HPV-31, HPV-33, HPV-35, HPV-39, HPV-52,
HPV-56, HPV-58, HPV-59, HPV-26, HPV-53, and HPV-66.

19. The method of claim wherein each of the HPV probes in the mixture
comprise a biotin label.

20. The method of claim 16 wherein the detecting of the presence of HPV
infection and the determination of the hybridization pattern of the
chromosomal probes is performed using digital imaging.

21. The method of claim 16 wherein presence of a hybridization pattern
indicative of the presence of cervical HSIL is identified in three or
more HPV infected cells.

22. The method of claim 20 wherein presence of a hybridization pattern
indicative of the presence of cervical HSIL is identified in one or more
HPV infected cells.

23. The method of claim 1, wherein the sample is contacted with two to not
more than four chromosomal probes.

Description:

BACKGROUND OF THE INVENTION

[0001]Cervical cancer remains one of the most common cancer types
affecting women worldwide. The biological pathway to cervical carcinoma
begins with normal intraepithelial cells, and develops through low and
then high grade dysplasia before malignancy obtains. Cytologists mark the
passage to malignancy as progression from normal epithelial cells to
atypical squamous cells of undetermined significance (ASCUS) to Low Grade
squamous intraepithelial lesions (LSIL) and then high grade squamous
intraepithelial lesions (HSIL) before carcinoma in situ and finally
malignancy result. Histologists mark the progression from normal cells to
various grades of cervical intraepithelial neoplasia (CIN I, II and III),
then to carcinoma in situ and finally malignancy. CIN I is considered low
grade dysplasia comparable to LSIL. CIN II and III are considered high
grade dysplasia comparable to HSIL.

[0002]The current standard of care includes regular cytologic testing with
a Papanicolau (Pap) smear to identify abnormalities as indicating
dysplasia or carcinoma in patient cells. When high grade dysplasia is
detected and confirmed by histological examination, the transformation
zone of the patient's cervix is removed immediately by loop excision or
cone biopsy. More radical procedures are required when carcinoma is
detected. At the same time, however, the progression from normal to
malignancy is not strict and the presence of low grade dysplasia does not
necessarily indicate that the patient will progress to high grade
dysplasia or malignancy. Significantly, the negative predictive value of
cytologic methods (e.g., Pap smears) for detecting high grade dysplasia
is poor. Thus, low grade dysplasia may be misdiagnosed as high-grade,
thereby subjecting the patient to unwarranted treatment and high grade
dysplasia may be misdiagnosed as low grade dysplasia, thereby delaying
appropriate treatment. Accordingly, there is a need for a diagnostic
method that will accurately distinguish between low and high grade
dysplasia.

[0003]Patient specimens typically comprise many thousands of cells for
evaluation. Diagnosis based on evaluation of individual cells can be
enormously time consuming and tedious for technicians to perform due to
the large number of cells that are required for evaluation. Thus, there
is a need for a means to simplify a cell evaluation method.

[0004]Others have noted that genetic abnormalities (e.g., changes in
chromosome regions or changes in ploidy levels) accompany the progression
from normal cells to cervical malignancy. See, e.g., U.S. Pat. No.
5,919,624 to Ried, et al. Ried et al. noted that chromosomal
abnormalities can be used to classify the progression of dysplastic
cervical cells in late stages, e.g., from noninvasive cervical to
invasive cervical carcinoma. Still others have demonstrated that cervical
cancer is associated with infection by certain human papilloma viruses
(HPV) types, particularly HPV types 16, 18, 31, 33, 35 and 42. See, e.g.,
Lazo, Brit. J. Cancer, (1999) 80(12), 2008-2018. Additionally, many cell
cycle proteins such as p16 and Cyclin E and cell proliferation markers
such as the proteins Ki67 and PCNA are also known to be highly active in
neoplastic cells. Thus, cells containing abnormal amounts of these
markers have been suggested as good candidates for cells that may
progress to malignancy.

[0005]PCT application WO 0024760 describes methods and reagents for
detecting HPV DNA in Pap smears using in situ hybridization and
brightfield microscopy. The probe consists of full length DNA probes of
HPV-16, -18, -31, -33, -35, and -51. The patent claims that this probe
mix detects other high-risk BPV types but not low-risk HPV. The ability
of the disclosed HPV probe mixture to avoid hybridization to low-risk BPV
types is achieved by modulation of the quantities of each HPV DNA probe
included on the probe mix. The BPV probes disclosed are different than
those described herein. In addition, the assays of the invention modulate
probe cross-hybridization by lowering the stringency of the hybridization
conditions while keeping the probe concentrations constant for all types.
This application also does not combine HPV probe with use of chromosomal
probes to detect chromosome abnormalities in the HPV infected cells.

[0006]Hopman et al. (J of Pathology 2004; 202:23-33) analyzed HPV status
and chromosomal aberrations in cervical biopsies sections by FISH. This
work used only probes for HPV-16 and BPV-18 and genomic probes for
chromosome 1 (1q12), 17, and X. In contrast to the inventive assay that
simultaneously detect HPV and chromosomal gains in the same cells, Hopman
et al.'s detection of HPV positive cells and chromosomal aberrations was
performed in parallel tissue sections.

[0007]To date Applicants are not aware of any publication that has
demonstrated that any chromosomal abnormality with or without the
presence of another marker can be used to distinguish low from high grade
dysplasia or has combined such a diagnostic method with the known
association of HPV and cervical cancer.

SUMMARY OF THE INVENTION

[0008]The invention is based on the discovery that certain chromosomal
abnormalities can be used to selectively detect high grade cervical
intraepithelial neoplasia (CIN II and CIN III) and malignant carcinoma in
cervical biopsy and Pap smear specimens without detecting low grade
cervical intraepithelial neoplasia. The method can detect high grade
cervical intraepithelial neoplasia (CIN II and CIN III) and malignant
carcinoma at high sensitivity and specificity levels, i.e. about 95%
each. The invention is based on the use of in situ hybridization
technology where labeled nucleic acid probes are allowed to hybridize to
cervical samples. Preferably, fluorescent in situ hybridization (FISH) is
used and the nucleic acid probes are DNA probes that are fluorescently
labeled. The hybridization results are then correlated with a clinical
diagnosis of high grade cervical intraepithelial neoplasia (CIN II and
CIN III) and malignant carcinoma. The method of the invention utilizes a
set of one or more probes demonstrating a vector value for discriminating
between CIN I and CIN II of about 60 or less, wherein the vector value is
calculated by
Vector=[(100-specificity)2+(100-sensitivity)2]1/2.
Preferred probes for use in the method are probes to the genetic loci
3q26, 8q24, 20q13, Xp22 and 3p21, and probes that enumerate chromosomes 3
and 15. Multiple probe sets comprising two, three or more probes can be
used in the method of the invention. Preferred multiprobe sets comprise
probes to the genetic loci 8q24 and 3q26; 3q26, 8q24, Xp22, and
chromosome 15; 8q24, 20q13, Xp22 and chromosome 15; and the genetic loci
3p21, 3 p14, 3q26 and chromosome 3. Probes useful in the invention can be
incorporated into kits packaged, for example, with other reagents useful
in carrying out the methods of the invention. Such kits can comprise one
or more probes useful with the invention.

[0009]Probes can be selected using the steps of: (a) providing a first
plurality of chromosomal probes (by plurality is meant one or more
probes); (b) determining the ability of each of the first plurality of
probes to distinguish high (CIN II, CIN III and carcinoma) from low (CIN
I) grade dysplasia in a cervical specimen; and (c) selecting the probe or
probes within the first plurality of probes that distinguish high from
low grade dysplasia to yield a second plurality of probes, wherein the
second plurality of probes identifies the high grade dysplasia specimens
as compared to low grade specimens at a vector value of less than about
60. Preferred probes can be selected by additionally: (d) determining the
ability of a combination of probes selected from the second plurality of
probes to distinguish the high grade from low grade specimens; and (e)
selecting a combination of probes that identifies the high grade specimen
as compared to the low grade specimen with a vector value of less than
about 40. More preferred embodiments can be selected based on lower
vector values (e.g., a vector value of less than about 30). The
biological sample used with the invention can contain a cervical biopsy
specimen or a cervical smear such as a Pap smear or a ThinPrep®
sample prepared by the method of Cytyc Corp., Boxborough, Mass. The
probes used with the invention comprise detectably labeled nucleic
acid-based probes, such as deoxyribonucleic acid (DNA) probes or protein
nucleic acid (PNA) probes, which are desiped/selected to hybridize to the
specific designed chromosomal target. Fluorescent labels such as are used
in fluorescent in situ hybridization are preferred but other detectable
labels commonly used in hybridization techniques, e.g., enzymatic,
chromogenic and isotopic labels, can also be used.

[0010]In another aspect of the invention, the detection of the genetic
abnormalities is facilitated by adoption of a preliminary cell screening
technique whereby cervical cells are screened first for the presence of a
suitable associated marker, for example, such as the presence of
infection by EPV, e.g., high risk HPV, or abnormal amounts of cell cycle
proteins such as p16 and Cyclin E or cell proliferation markers such as
Ki67 and PCNA. Such screening can be used to identify more suspicious
cells for closer examination and may allow the time required for specimen
evaluation to be reduced by as much as 5-10 fold. After the suspicious
cells are identified, these suspicious cells are then examined for the
presence of chromosomal abnormalities. The presence of chromosomal
abnormalities identified by use of the probes of the invention in cells
also showing markers of potential malignancy, such as HPV infection,
identifies higher grade CIN or malignancy. Such initial screening
techniques are amenable to automation, enabling greater simplicity and
speed in specimen evaluation.

[0011]A preferred assay comprises the simultaneous detection of HPV
infection and chromosomal gains by fluorescence in situ hybridization in
individual cells on cervical cytological specimens to identify higher
grade disease. This preferred assay comprises a method for screening for
high grade dysplasia in a subject, the method comprising: (a) obtaining a
biological sample from the subject; (b) contacting the sample with a set
of one or more chromosomal probes and with a mixture of HPV probes under
conditions sufficient to enable hybridization of the probes to
chromosomes in the sample if any and sufficient to enable detection of
HPV infected cells present in the sample if any; (c) detecting the
presence of HPV infected cells in the sample; and (d) determining
hybridization pattern of the chromosomal probes in the BPV infected cells
in the sample to determine whether the subject has high grade dysplasia.
Detection of HPV is preferably done with a mixture of six HPV full-length
genomic probes (HPV-16, BPV-18, HPV-30, HPV-45, HPV-51, and HPV-58) under
low stringency hybridization conditions. Use of this mixture under low
stringency conditions will detect the following HPV types: HPV-16,
HPV-18, HPV-31, HPV-33, HPV-35, HPV-39, HPV-45, HPV-51, HPV-52, BPV-56,
HPV-58, BPV-59, HPV-26, HPV-53, and HPV-66. Preferably, the six HPV
probes in the mixture are labeled so that the probes are detected using a
fluorescence labeled tyramide signal amplification-system. Detection of
chromosomal gains preferably is done with three directly labeled probes,
each labeled in a fluorescent color distinct from the others and from the
HPV cocktail detection color: to chromosomal locus 8q24 and 3q26 and to
the centromere of chromosome 8. The determination of a hybridization
pattern indicative of the presence of chromosomal abnormalities in cells
infected with high-risk HPV correlates with high-grade dysplasia.

DETAILED DESCRIPTION OF THE INVENTION

[0012]The invention includes (i) methods of using probes and (ii) probe
sets for the detection of high grade dysplasia and carcinoma in cervical
cells. The methods and probe sets allow for the early detection of high
grade dysplasia in biological samples, such as a cervical biopsies and
smears.

Chromosomal Probes

[0013]Suitable probes for use in the in situ hybridization methods
utilized with the invention fall into two broad groups: chromosome
enumeration probes, i.e., probes that hybridize to a chromosomal region,
usually a repeat sequence region, and indicate the presence or absence of
an entire chromosome, and locus specific probes, i.e., probes that
hybridize to a specific locus on a chromosome and detect the presence or
absence of a specific locus. Chromosome arm probes, i.e., probes that
hybridize to a chromosomal region and indicate the presence or absence of
an arm of a specific chromosome, may also be useful. Chromosomal probes
and combinations thereof are chosen for sensitivity and/or specificity
when used in methods of the invention. Probe sets can comprise any number
of probes, e.g., 1, 2, 3, 4 or more probes. The number of probes useful
with the invention is limited only by the user's ability to detect the
probes on an individual basis.

[0014]As is well known in the art, a chromosome enumeration probe can
hybridize to a repetitive sequence, located either near or removed from a
centromere, or can hybridize to a unique sequence located at any position
on a chromosome. For example, a chromosome enumeration probe can
hybridize with repetitive DNA associated with the centromere of a
chromosome. Centromeres of primate chromosomes contain a complex family
of long tandem repeats of DNA comprised of a monomer repeat length of
about 171 base pairs, that are referred to as alpha-satellite DNA.
Non-limiting examples of chromosome enumeration probes include probes to
chromosomes 1, 6, 7, 8, 9, 10, 11, 12, 15, 16, 17, 18 and X. Examples of
several specific chromosome enumeration probes are described in Example
1.

[0015]A locus specific probe hybridizes to a specific, non-repetitive
locus on a chromosome. Non-limiting examples of locus specific probes
include probes to the following loci: 3q26, 8q24, 20q13, Xp22 and 3p21.
Some of these loci comprise genes, e.g., oncogenes and tumor suppressor
genes that are altered in some forms of cervical cancer. Thus, probes
that target these genes, either exons, introns, or regulatory chromosomal
sequences of the genes, can be used in the detection methods described
herein. Examples of target genes include: TERC (3q26); MYC (8q24); STK6
(20q13.2-13.3) and MLH (3p21-p23). Additional examples are identified in
Example 1.

[0016]Probes that hybridize with centromeric DNA and specific chromosomal
loci are available commercially from Vysis, Inc. (Downers Grove, Ill.)
and Molecular Probes, Inc. (Eugene, Oreg.). Alternatively, probes can be
made non-commercially using well known techniques. Sources of DNA for use
in constructing DNA probes include genomic DNA, cloned DNA sequences such
as bacterial artificial chromosomes (BAC), somatic cell hybrids that
contain one or a part of a human chromosome along with the normal
chromosome complement of the host, and chromosomes purified by flow
cytometry or microdissection. The region of interest can be isolated
through cloning or by site-specific amplification via the polymerase
chain reaction PCR). See, for example, Nath, et al., Biotechnic
Histochem, 1998, 73 (1): 6-22; Wheeless, et al., Cytometry, 1994,
17:319-327; and U.S. Pat. No. 5,491,224. Synthesized oligomeric DNA or
PNA probes can also be used.

[0017]The size of the chromosomal region detected by the probes used in
the invention can vary, for example, from the alpha satellite 171 base
pair probe sequence noted above to a large segment of 150,000 bases. For
locus-specific probes, that are directly labeled, it is preferred to use
probes of at least 100,000 bases in complexity, and to use unlabeled
blocking nucleic acid, as disclosed in U.S. Pat. No. 5,756,696, herein
incorporated by reference, to avoid non-specific binding of the probe. It
is also possible to use unlabeled, synthesized oligomeric nucleic acid or
protein nucleic acid as the blocking nucleic acid. For targeting a
particular gene locus, it is preferred that the probes span the entire
genomic coding locus of the gene.

[0018]Chromosomal probes can contain any detection moiety that facilitates
the detection of the probe when hybridized to a chromosome. Effective
detection moieties include both direct and indirect labels as described
below.

[0021]When multiple probes are used, fluorophores of different colors can
be chosen such that each chromosomal probe in the set can be distinctly
visualized. Preferably the probe panel of the invention will comprise
four separate probes, each labeled with a separate fluorophore. Use of
four probes is preferred because Applicants believe this provides the
best balance between clinical sensitivity (sensitivity can increase with
added probes) and imaging/detection complexity (complexity can increase
with added probes). It is also within the scope of the invention to use
multiple panels sequentially on the same sample: in this embodiment,
after the first panel is hybridized, the results are imaged digitally,
the sample is destained and then is hybridized with a second panel.

[0022]Probes can be viewed with a fluorescence microscope and an
appropriate filter for each fluorophore, or by using dual or triple
band-pass filter sets to observe multiple fluorophores. See, e.g., U.S.
Pat. No. 5,776,688 to Bittner, et al., which is incorporated herein by
reference. Any suitable microscopic imaging method can be used to
visualize the hybridized probes, including automated digital imaging
systems, such as those available from MetaSystems or Applied Imaging.
Alternatively, techniques such as flow cytometry can be used to examine
the hybridization pattern of the chromosomal probes.

[0023]Probes can also be labeled indirectly, e.g., with biotin or
digoxygenin by means well known in the art. However, secondary detection
molecules or further processing are then required to visualize the
labeled probes. For example, a probe labeled with biotin can be detected
by avidin conjugated to a detectable marker, e.g., a fluorophore.
Additionally, avidin can be conjugated to an enzymatic marker such as
alkaline phosphatase or horseradish peroxidase. Such enzymatic markers
can be detected in standard colorimetric reactions using a substrate for
the enzyme. Substrates for alkaline phosphatase include
5-bromo-4-chloro-3-indolylphosphate and nitro blue tetrazolium.
Diaminobenzoate can be used as a substrate for horseradish peroxidase.
Fluorescence detection of a hybridized biotin or other indirect labeled
probe can be achieved by use of the commercially available tyramide
amplification system.

[0024]Detection of HPV can be done using one or more probes comprising the
entire genomic sequence, of an BPV type or a partial genomic sequence,
such as a mixture of whole genomic probes to HPV types 16 and 18. The HPV
probe mixture used should be sufficient to identify the presence of the
major high risk types, including HPV-16, HPV-18, BPV-31, HPV-33, HPV-35,
HPV-39, HPV-45, HPV-51, HPV-52, BPV-56, HPV-58, BPV-59, HPV-26, HPV-53,
and HPV-66. A preferred mixture comprises six full-length PV genomic
probes (HPV-16, HPV-18, HPV-30, HPV-45, HPV-51, and HPV-58) which is used
under low stringency hybridization conditions. These six probes were
selected based on sequence homology analysis with other high-risk HPV
types. Based on sequence homology and on the assumption that HPV types
with 50% or higher homology to these six HPV types will show
cross-hybridization, use of this preferred mixture under low stringency
conditions will detect the following HPV types: HPV-16, HPV-18, HPV-31,
HPV-33, HPV-35, HPV-39, HPV-45, HPV-51, HPV-52, HPV-56, HPV-58, HPV-59,
BPV-26, HPV-53, and HPV-66. In this preferred mixture, the concentrations
of each of the six HPV probes is maintained at approximately equal
amounts, which is preferably less than a 5 percent difference in the
individual probe amounts by weight. Preferably, the six HPV probes in the
mixture are labeled so that the probes are detected using a fluorescence
labeled tyramide signal amplification system.

[0025]The probes and probe sets useful with the methods of the invention
can be packaged with other reagents into kits to be used in carrying out
the methods of the invention. Useful kits can comprise one or more probes
from the group of probes to the genetic loci 3q26, 8q24, 20q13, Xp22 and
3p21, and probes that enumerate chromosomes 3 and 15. A preferred kit of
the invention comprises four probes: (i) a biotin labeled mixture of six
HPV probes (for HPV types 16, 18, 30, 45, 51 and 58); (ii) a chromosomal
probe to TERC gene locus at 3q26; (iii) a chromosomal probe to the cmyc
gene locus at 8q24; and (iv) a chromosomal probe to the centromere of
chromosome 8.

Determining the Presence of High Grade Dysplasia

Pre-Selection of Cells

[0026]Cell samples can be evaluated preliminarily by a variety of methods
and using a variety of criteria. The probes and methods described herein
are not limited to usage with a particular screening methodology. One
example is the "scanning method" wherein the observer scans hundreds to
thousands of cells for cytologic abnormalities, e.g., as viewed with a
DAPI filter. The number of cells assessed will depend on the cellularity
of the specimen, which varies from patient to patient. Cytologic
abnormalities commonly but not invariably associated with dysplastic and
neoplastic cells include nuclear enlargement, nuclear irregularity, and
abnormal DAPI staining (frequently mottled and lighter in color). In the
scanning step, the observer preferably focuses the evaluation of the
cells for chromosomal abnormalities (as demonstrated by FISH) to those
cells that also exhibit cytological abnormalities. In addition, a
proportion of the cells that do not have obvious cytologic abnormalities
can be evaluated since chromosomal abnormalities also occur in the
absence of cytologic abnormalities. This scanning method is described in
further detail in U.S. Pat. No. 6,174,681 to Halling, et al., which is
incorporated herein by reference.

[0027]More preferably, the observer can scan the cells for a marker
associated with cancer. For example, the cells can be scanned for the
presence of an associated marker such as the presence of BPV or high risk
HPV (e.g., one or more of PV types 16, 18, 31, 33, 35 or 45).
Additionally, cells with abnormal amounts of the cell cycle proteins p16
and Cyclin E or the proliferation markers Ki67 and PCNA are likely to be
suspicious and good candidates for closer examination. Cells can be
scanned for the presence of these markers using well know methods. Cell
scanning is generally amenable to automation. Automated scanning permits
increased efficiency by permitting assays to be performed more rapidly
and eliminating much of the tedium present in manual scanning.

Preparation of Samples

[0028]The presence or absence of high grade dysplasia and carcinoma can be
determined by identifying chromosomal aberration in the cells. This can
be accomplished by in situ hybridization. In general, in situ
hybridization includes the steps of fixing a biological sample,
hybridizing a chromosomal probe to target DNA contained within the fixed
sample, washing to remove non-specifically bound probe, and detecting the
hybridized probe. The in situ hybridization can also be carried out with
the specimen cells in liquid suspension, followed by detection by flow
cytometry.

[0029]Abnormal cells are characterized by abnormal numbers of chromosomes
within the cells and/or structural alterations within the cells'
chromosomes. Structural alterations can include gains or losses (e.g.,
hemizygous or homozygous loss) of a specific chromosomal region, such as
a locus or centromeric region as indicated in Example 1. Positive test
indicators can be developed accordingly. For example, a cell having one
or more chromosomal gains, i.e., three or more copies of any given
chromosome, can be considered to test positive in the methods described
herein. Cells exhibiting monosomy or nullisomy may also be considered
test positive under certain circumstances.

[0030]A biological sample is a sample that contains cells or cellular
material, e.g., cells or material derived from the uterine cervix of the
uterus. Examples of cervical specimens include cervical biopsies, smears,
scrapes and the like. Typically, cells are harvested from a biological
sample and prepared using techniques well known in the art. Numerous
methods are available for collecting cervical cells for evaluation. For
example, cells from the ectocervix and endocervix/transformation zone are
collected using well-known devices such as endocervical brushes (or
"brooms") or wooden and plastic spatulas. Conventional smears are
prepared by spreading cells evenly and thinly onto a glass slide. The
slide is then fixed rapidly by immersion into 95% ethanol or spraying
with a commercial fixative according to manufacturer instructions. For
the ThinPrep® collection method (Cytyc Corp., Boxborough, Mass.),
cells are transferred from the cervix into the fixative PreservCyt®.
This allows cells to be preserved until ready for further processing.
Cells are then gently dispersed, randomized and collected onto a
TransCyt® membrane filter by drawing the sample across the filter
with a vacuum until an optimal number of cells is deposited into the
filter. The cells can be further processed as desirable. In another
method, the cells collected into PreservCyt® or other fixative
solution can be further washed by centrifuging, removing the supernatant
and resuspending in Carnoys solution (3:1 Methanol:Acetic acid),
repeating (e.g., three times) as desired. Cells are then transferred to a
glass slide by dropping a small aliquot of cell suspension directly onto
the slide. Slides are typically dried overnight.

Detection of Chromosomal Abnormalities

[0031]Gain or loss of chromosomes or chromosomal regions within a cell is
assessed by examining the hybridization pattern of the chromosomal probe
or set of chromosomal probes (e.g., the number of signals for each probe)
in the cell, and recording the number of signals. Test samples can
comprise any number of cells that is sufficient for a clinical diagnosis,
and typically contain at least about 100 cells. In a typical assay, the
hybridization pattern is assessed in about 25-5,000 cells. Test samples
are typically considered "test positive" when found to contain a
plurality of chromosomal abnormalities, e.g., cells present gains or
losses of one or more chromosomes, loci or chromosomal arms as described
herein. Criteria for "test positive" can include testing positive with
one, two, three, four or more probes. Testing positive with one probe is
a typical test criterion; testing positive with two probes is more
preferred, and with four is most preferred. In addition, when multiple
probes are used test positive can include detection of abnormal
hybridization patterns with a subset of probes, e.g., a combination of
gains or losses of a subset of the probes, e.g., two or three probes of a
full set of four probes. Hybridization patterns can be assessed in
sequence for subsets of probes. For example, the pattern of an initial
subset of probes (e.g., probes to the 3q26 and 8q24 loci) can be assessed
and, if a positive result is indicated from the subset of probes the test
can be taken as positive overall. However, if the initial result is not
positive, the pattern for an additional subset of probes (e.g., probes to
the Xp22 locus and chromosome 15) can be assessed to complete the test.
If the combined result for all probes indicates a positive test result,
the test can be taken as positive overall. The number of cells identified
with chromosomal abnormalities and used to classify a particular sample
as positive, in general will vary with the number of cells in the sample.
As low as one cell may be sufficient to classify a sample as positive. It
is preferred to identify at least 30 cells as positive, more preferred to
identify at least 10 cells, and most preferred to identify at least 5
cells as positive. The number of cells used for a positive classification
is also known as the cut-off value, which is discussed further below.

Screening and Monitoring Patients for High Grade Dysplasia and Cervical
Carcinoma

[0032]The methods described herein can be used to screen women for high
grade dysplasia as a predecessor to cervical carcinoma. For example,
women at risk for cervical cancer, e.g., women with abnormal PAP smear,
women who are infected with a HPV, e.g., high risk HPV, or women that
show abnormal amounts of cell cycle proteins such as p16 and Cyclin E or
cell proliferation proteins such as Ki67 and PCNA can be regularly
screened with the goal of early detection of progression to high grade
dysplasia. For example, general probes and methods to detect infection by
HPV in a sample can be used, such as, for example, a whole genomic HPV
probe. Type specific probes can also be developed to detect infection by
specific HPV types such as one or more of the high risk HPV types HPV 16,
18, 31, 33, 35, 45, 51, 52 and 58. Alternatively, antibodies are know and
can be adapted to detect the presence of specific proteins such as the
p16 and Ki67 proteins in a sample. In this embodiment, the sample is
first assayed with the HPV probe or the antibody probe to identify
particular cells. The labeled cells are then assessed as to the
chromosomal status using a probe panel of the invention. The HPV or
antibody step can be performed simultaneously or sequentially with the
chromosomal probe panel.

[0033]The screening test can be incorporated into the routine care of
women, e.g., as an adjunct to evaluation of routine Pap smears. The
methods described here can also be used to adjust treatment strategies
for women. As a more reliable test than the conventional tests, e.g., Pap
tests, patients can be directed more reliably to the invasive remediation
(removal of the transformation zone of the patient's cervix) as
necessary. Patients testing negative for high grade dysplasia by the test
methodology can be spared this invasive procedure more reliably.

Probe Selection Methods

[0034]The selection of individual probes and probe sets for use with the
invention can be performed using the principles described in the
examples. Each probe selected for a probe set should have the ability on
its own to discriminate between high and low grade dysplastic cells.
Probes with high discrimination ability are preferred. The discrimination
analysis described herein comprises calculating the sensitivity and
specificity of each probe individually for identifying high and low grade
dysplasia. Various cutoff values of cell percentages for targets gained
and lost are employed. The primary metric for combined sensitivity and
specificity will be a quantity called `vector`, which is defined as the
magnitude of the vector drawn between the points on a sensitivity versus
specificity plot representing the ideal (sensitivity=specificity=100) and
the measured sensitivity and specificity of the particular probe or probe
set, as measured in a cohort of abnormal and normal samples. As described
in Example 2, the vector value ranges from 0 for the ideal case to 141.4
for the worst case. Statistical analyses can also be used to compare
means and standard deviations between high and low grade dysplastic cells
as described in U.S. patent application Ser. No. 10/081,393 by Morrison,
et al. filed Feb. 20, 2002, which is incorporated herein by reference.

[0035]For multiple probes sets, each probe should be selected to
complement the other probes in the set. That is, each probe should
identify additional high grade dysplasia markers that the other probe(s)
fail to identify. One method for identifying the best complementing set
of probes is to take the probe with the lowest vector value, remove the
group of tumor specimens it identified from the full set of tumor
specimens, and then determine the probe with lowest vector value on the
remaining tumor specimens. This process can be continued as necessary to
obtain a complete probe set. The approach described here of generating
all possible probe combinations, and calculating the sensitivity and
specificity of each, predicts the performance of all possible probe sets
and allows selection of the minimal probe set with the highest
performance characteristics. Also, a variety of combinations with
similarly high performance characteristics is obtained. Considering the
possible errors due to the finite number of specimens tested, several of
the high ranking probe combinations can be compared based on other
practical characteristics such as relevance to disease prognosis or
difficulty in making the probe.

[0036]However, regardless of the measured ability to complement other
probes, each probe must preferably identify a statistically different
percentage of test positive cells between the high and low grade adjacent
specimen sets. If this condition is not met, then a probe might be
selected erroneously based on apparent complementation. Moreover, data
from combinations of fewer probes is more reliable than data from
combinations of more probes, e.g., data from combinations of two probes
is more reliable than data from combinations of three probes. This
results from the reduced ability to make correlations between greater
numbers of probes with the finite number of specimens tested.

[0037]The dependence of probe and probe combination performance as a
function of cutoff value must also be considered. "Cutoff value" can
refer to the number or percentage of cells in a population that must have
gains or losses for the sample to be considered positive. Therefore, a
sample can be considered positive or negative depending upon whether the
number (or percentage) of cells in the specimen is above the cutoff value
or equal to or less than the cutoff value, respectively. In general, the
combined specificity and sensitivity of probes is better at low cutoff
values. However, when the high grade dysplasia cells are distributed
within a matrix containing many normal and low grade cells, such as from
a cervical smear, probes performing best at high cutoffs are more likely
to be detected. This is because good performance at high cutoffs
indicates a higher prevalence of cells containing the abnormality.
Examples of cutoff values that can be used in the determinations include
about 5, 25, 50, 100 and 250 cells or 5%, 10%, 15%, 20%, 25%, 30%, 35%,
40%, 50% and 60% of cells in the sample population. In the preferred
assay combining identification of HPV infected cells and determination of
chromosomal abnormalities present in the HPV infected cells, a cutoff
value of three (3) positive cells is preferred.

[0038]Measurement of gain of a target chromosome or chromosome region is
preferred over measurement of a target chromosome or chromosome region
loss, because overlapping targets or poor/failed hybridization to some
cells can falsely suggest loss. Locus-specific or chromosomal arm probes
designed to detect deletions are also generally smaller than
locus-specific or chromosomal arm probes designed to detect gains since
the deletion probes must not extend beyond the minimally deleted region.
If too much of the "deletion probe" extends beyond the deleted sequence,
enough signal may be produced in the assay to be falsely counted. Since
"deletion probes" are usually kept small their signals are not as intense
as signals for targets typically gained. This in turn makes it more
likely that real signals from targets being monitored for deletion may be
miscounted. Likewise, repetitive sequence probes, like some chromosome
enumeration probes used here are preferable to single locus probes
because they usually provide brighter signals and hybridize faster than
locus specific probes. On the other hand, repetitive sequence probes are
more sensitive to polymorphisms than locus specific probes.

[0039]A probe or combination of probes used with the present invention
preferably provides an improvement over conventional methods such as
cytology. Useful probes or probe combinations of the invention identify
at least about 70% and preferably above about 85% of samples with high
grade dysplasia and carcinoma (sensitivity). Similarly, useful probes or
probe combinations identify as negative by test at least about 80% and
preferably above about 95% of negative samples (specificity).

[0040]The invention is, further described in the following examples, which
are not intended to limit the scope of the invention described in the
claims.

EXAMPLES

[0041]1. Initial Probe Selection. Thirty-five chromosomal regions,
identified in Table 1 below, known to show some level of amplification or
deletion in cervical cancer or dysplasia were selected for evaluation.
The colors in Table 1 refer to the fluorescent label used for each of
these probes.

[0042]Thirteen of these regions were detected using chromosome enumeration
probes (CEP® probes in Table 1)) targeting repetitive centromeric
sequences. Twelve of the CEP® probes used are commercially available
from Vysis, Inc. (Downers Grove, Ill.). The other twenty-two regions were
detected with locus specific probes targeting unique sequences within
amplified or deleted chromosomal regions.

[0043]Seven of these locus specific probes used are commercially
available, labeled with SpectrumOrange® label, from Vysis, Inc.
(marked with an asterisk in Table 1.) The commercial STS probe was used.
For the other six probes, the same starting DNA material as used to make
the commercially available probes was used. Instead of the SpectrumOrange
label, the starting DNA was transaminated and then chemically labeled
using 5-(and -6)-carboxyrhodamine 6G, succinimidyl ester (molecular
Probes) for the c-myc and CCND1 probes, and fluorescin succinimidyl ester
for the other four. The transamination and labeling process used is
described in Bittner et al., U.S. Pat. No. 5,491,224, incorporated herein
by reference. The CEP 11 probe labeled in gold was produced using the
starting DNA material of the commercially available CEP 11 probe from
Vysis, Inc., and using the same procedure as for the c-myc probe.

[0044]The remaining 14 probes were produced from BAC clones sourced as
shown in Table 2.

[0045]The HER-4, FHIT, DDX15 and DAB2 probes were also produced using the
same method as the c-myc probe. The remaining unique sequence probes were
all produced using the nick translation method described in Morrison
2002, Id. at p. 27-30, and the labeled nucleotides Spectrum Orange dUTP,
Spectru Red dUTP or Spectrum Green dUTP (all Vysis, Inc.).

[0046]The labeled probes were then separated into sets of three or four
probes each for evaluation as indicated in Table 1. The probe sets were
made up of the individual probes, COT1 DNA (Invitrogen), human placental
DNA (Signa), and LSI/WCP Hybridization Buffer (Vysis, Inc.). 10 μl of
each of the probe sets were hybridized to ten samples each of cervical
biopsy samples. The probe sets each typically contained about 0.5 μg
COT1 DNA and 2 μg human placental DNA. The probe set hybridization
mixes also contained 50 nanograms of Spect Aqua labeled human placental
DNA to provide a background staining of the nuclei in the sample, as
described in U.S. Pat. No. 5,789,161, Morrison et al., incorporated
herein by reference. CIN I, CIN II-III and invasive cervical squamous
carcinoma (CA) samples were obtained from the Cooperative Human Tissue
Network (CHTN) supported by the National Cancer Institute. The samples
were prepared for hybridization and hybridized with the probe sets as
follows. Paraffin embedded tissue sections were placed in xylene solution
for 5 min. This procedure was repeated 3 times. Slides were then washed
in 100% ethanol twice for 1 min each wash. Slides were then soaked for 15
min in 45%/0.3% peroxide solution, rinsed in water and incubated for 10
min in Pretreatment solution. After rinsing, slides were incubated with a
proteinase, e.g., proteinase K or pepsin, for 5-30 min to digest excess
proteins and make the DNA more accessible. The slides were then
dehydrated in ethanol series, air dried and hybridized with DNA probes
usually overnight at 37° C. After hybridization, unspecific probes
were washed out in post-hybridization wash solutions such as for example,
wash for 2 minutes in 73±1° C. 2×SSC/0.3% NP40. Slides
were then washed in a second wash solution such as 2×SSC/0.1% NP40.
A DAPI DNA stain was then applied to the slides to facilitate sample
evaluation.

[0047]The procedure permitted all probes to hybridize to the samples. The
majority of probes showed good signal intensity relative to background.
The epithelial layers of the biopsy samples were evaluated under a
fluorescence microscope to identify any cells that showed amplification
(more than two signals) or deletion (less than two signals) of the DNA
target. Gains were recorded for each sample that showed amplification in
five or more cells for a particular probe; losses were recorded for each
sample that showed a deletion in five or more cells. Samples showing
neither gains nor losses were considered disomic.

[0048]The sensitivity, i.e., the percentage of samples showing the
condition tested, of each probe for CIN I, CIN II-III and invasive
carcinoma was determined for gains, losses and disomies. Losses were
found to occur very infrequently in CIN II-III samples and so were not
generally useful as markers for CIN II-III and invasive carcinoma. Probes
were further assessed for their ability to show maximum frequency of
gains for CIN II-III and minimum frequency of gains for CIN I. The
results are presented in Table 3. Probes for the targets 8q24, 20q13,
3p21, 3q26, 1p31, Xp22 and CEP 15 were considered the most informative
and were selected for further evaluation. The 3 p14 probe showed
significant loss in the CIN II-III and invasive carcinoma samples. The
ratio of the relative gain of 3q26 to 3 p14 was also evaluated as a
measure of the relative gain of the q arm of chromosome 3 to its p arm.

[0049]2. Discriminate Analysis of in Situ Hybridization Data and Selection
of Probe Sets.

[0050]Additional paraffin embedded biopsy samples classed as normal (WNL),
CIN I, CIN II, CIN III and Squamous cell carcinoma (CA) were obtained
from the University of Texas Southwestern Medical Center, Dallas, Tex.
(Dr. Raheela Ashfaq). The samples were prepared and hybridized to two
sets of probes (CEP 15, 8q24, Xp22 and 20q13; and CEP 3, 3q26, 3q14 and
3p21) as before. Six of the probes used--8q24, Xp22, CEP 15, 20q13, 3p21
and 3q26--were taken from the preferred probes identified in Example 1.
Two others--CEP 3 and 3p14--were compared with probes to 3p21 and 3q26 to
better assess the relationship of chromosome 3 in the progression to
cervical cancer.

[0051]The ability of individual probes and certain probe combinations to
discriminate between high and low grade dysplasia in cervical cells was
evaluated by determining the number of specimens correctly identified by
each probe or probe set. A cutoff number of five cells with gains or
losses was used to evaluate samples. A sample was called positive or
negative for high grade dysplasia or carcinoma depending upon whether the
number of cells in the sample was above the cutoff value or equal to or
less than the cutoff value, respectively. The accuracies of identifying
the positive samples (sensitivity) and negative samples (specificity)
were then used to select the best probes and probe combinations. Table 3a
lists the specificity and sensitivity of gain and loss for certain probe
targets.

[0052]The ability to discriminate between cellular types depends on the
overall specificity and sensitivity. Good discrimination requires good
specificity and sensitivity. Table 3b presents results for a combined
measure of specificity and sensitivity designated "vector". Vector is
calculated as

Vector=[(100-specificity)2+(100-sensitivity)2]1/2

Specificity and sensitivity are defined as percentages and range from 100%
(perfect) to 0% for no specificity (or sensitivity) at all. Hence, vector
values range from 0 for perfect specificity and sensitivity to 141 for
zero specificity and sensitivity.

[0053]Table 3b is sorted by increasing vector value for each sample
category. Individual probes showing a high ability to discriminate (low
vector value) include 3q26, 8q24 and CEP 3. Other probes showing a useful
ability to discriminate high grade dysplasia and carcinoma from low grade
dysplasia are 20q13, Xp22, CEP 15 and 3p21. Vectors determined for probe
ratios such as 3q26/CEP (determined to be >1) and 3q26/3 p14
(determined to be >1) can also be useful. Other methods for evaluating
and selecting probes using discriminate and combinatorial analytical
techniques are described in U.S. Ser. No. 10/081,393 by Morrison, et al.,
filed Feb. 20, 2002, which is incorporated herein by reference.

[0054]Based on results from discriminate analysis and probe
complementarity as described above, preferred probes for use in
distinguishing high from low grade dysplasia in cervical samples include
probes to the loci 3q26 and 8q24 and the CEP 3. Sets of probes comprising
the probes 3q26 and 8q24; 3q26, 8q24, Xp22, and CEP 15; 8q24, 20q13, Xp22
and CEP 15; and 3p21, 3p14, 3q26 and CEP 3 are particularly preferred.

[0055]3. Combined HPV and Chromosomal Gain Assay. A fluorescence in situ
hybridization assay to detect the presence of BPV infection and
chromosomal gains in the same cell(s) was developed and tested.

[0056]HPV plasmids and Probe composition Plasmids containing the full
coding sequence of HPV-16, HPV-18, HPV-30, BPV-45, HPV-51 and HPV-58 were
used to generate biotin labeled DNA probes by nick translation using a
convention protocol. HPV plasmids were obtained from the following
sources: HPV-16 and HPV-18 were purchased from the American Type Tissue
Collection (ATTC), BPV-30 and HPV-45 were obtained from Dr. Ethel-Michele
de Villiers (DKFZ, Germany), and HPV-51 and BPV-58 plasmids were obtained
from Klara Abravaya (Abbott Laboratories). The original sources for the
HPV-51 and HPV-58 plasmids are Dr. Saul Silverstein (Columbia University)
and Dr. Toshihiko Matsukura (Japan), respectively.

[0057]These six HPV probes were combined with locus specific 8q24 (cmyc)
and 3q26 (TERC) probes and the centromeric probe CEP-8. The locus
specific 8q24 and 3q26 probes used were those described above in Example
1., except that the 8q24 probe was labeled in Spectrum Red and the 3q26
probe was labeled in Spectrum Yellow using the method described in
Example 1.

[0059]Residual Thin-Prep preserved cervical specimens were obtained from
Mayo Clinic. Thin-Prep slides from the preserved cervical specimens were
prepared following the manufacturer's instructions (Cytyc). The samples
were prepared for hybridization as follows. Thin-Prep slides were soaked
in 2×SSC at 73° C. for 2 minutes, followed by incubation in
pepsin (0.5 mg/ml in 10 mM HCL) at 37° C. for 110 minutes. The
slides were then washed in 1×PBS at room temperature for 5 minutes,
fixed in 1% NBF (NBF-neutral buffer formalin) room temperature for five
minutes, and rinsed in 1×PBS at room temperature for 5 minutes.
After rinsing, the slides were dehydrated in ethanol series, air dried
and hybridized with the combined HPV and chromosomal probe mix overnight
at 37° C.

[0060]After hybridization, excess probes were washed in post-hybridization
wash consisting of 2×SSC/0.3% NP-40 for 2 minutes at 48° C.
and then 2×SSC/0.1% NP-40 for 1 minute at room temperature.

[0061]Detection of the Biotin labeled HPV probes was performed using the
Alexa Fluorm 488 Tyramide signal amplification kit (Molecular Probes)
following the manufacturer's directions. Briefly, endogenous peroxidase
activity was blocked by incubation in 3% H2O2 for 30 minutes at
room temperature and slides were washed in 1×PBS for 5 minutes at
room temperature. Slides were then incubated with 1% Blocking Reagent in
PBS at 37° C. for 25 minutes followed by Streptavidin-HRP at
37° C. for 25 minutes. After washing the slides 3 times in
1×PBS, the biotin labeled BPV probe/Streptavidin-HRP complex was
visualized by incubation with Alexa Fluor 488 labeled tyramide for
10ζ at room temperature. The slides were then washed in 1×PBS,
the nuclear counterstain DAPI was applied and slides were coverslipped.

[0062]Hybridized slides are analyzed under a fluorescence microscope. HPV
probe is visualized using the green filter and the staining could appear
as a diffuse staining throughout the cell nucleus, punctate staining or
mixed staining (punctuate and diffuse). Probe for Spectrum Yellow 3q26 is
detected using a gold filter, Spectrum Red 8q24 is detected using a red
filter, and Spectrum Aqua CEP8 is detected using an aqua filter. All
filters used are commercially available from Vysis, Inc., Downers Grove,
Ill.

HPV-Chromosomal Gain Assay Results

[0063]Residual Thin-Prep preserved cervical specimens were obtained from
Mayo Clinic. Fifty-seven specimens diagnosed with LSIL or HSIL cytology
were analyzed. After hybridization and washing as described above, the
slides were evaluated using fluorescence microscopy for the presence of
HPV and chromosomal gains. Results were correlated with available
histology and clinical follow up. Slide analysis was performed as follow:
(1) The whole surface area of the slide was analyzed using 40×
magnification to identify HPV positive cells, (2) For each positive cell
the BPV pattern (diffuse, punctate, or mix) and the chromosomal counts
for each probe were recorded, (3) In addition, chromosomal gains were
analyzed independent of the HPV status of the cell. Cells with 3 or more
signals for the MYC or TERC probe were recorded. The results are set out
in Tables 4 and 5.

[0064]As shown in Table 5, the combined HPV chromosomal abnormality assay
was able to identify high grade dysplasia at high sensitivity of 90%
using a cutoff value of three positive cells, while retaining acceptable
specificity.

Other Embodiments

[0065]While the invention had been described in conjunction with the
foregoing detailed description, it is to be understood that the foregoing
description is intended to illustrate and not limit the scope of the
invention. Other aspects, advantages and modification of the invention
are within the scope of the claims set forth below.